DU145 human prostate cancer cells express functional receptor activator of NFκB: New insights in the prostate cancer bone metastasis process
Introduction
Prostate cancer is the most common malignancy diagnosed in males and is currently the major leading cause of cancer death among men [1], [2]. Prostate cancer metastases to bone are observed in around 80% of prostate cancer patients and represent the most critical complication of advanced prostate cancer, frequently resulting in significant morbidity and mortality [1], [2]. Unlike other solid tumors that are associated with osteolytic bone metastases, prostate cancer bone metastases stimulate an overall increase in both bone remodelling and bone volume [3]. In contrast, recent findings strongly suggest the importance of osteoclast function [4], [5], [6], [7], [8], [9]; however the mechanisms underlining these processes are not fully determined. Therefore, understanding the biological mechanisms that govern prostate cancer metastases to bone at the molecular level and elucidation of the interactions among the factors involved should lead to the determination of new potential therapeutic targets.
Receptor activator of NFκB ligand (RANKL)/RANK/Osteoprotegerin (OPG) represent the key regulators of bone metabolism both in normal and pathological condition, including prostate cancer bone metastases [10], [11]. RANKL has been shown to both activate mature osteoclasts and mediate osteoclastogenesis in the presence of macrophage-colony stimulating factor (M-CSF) [12], [13]. RANKL is preferentially expressed on committed pre-osteoblastic cells, whereas its specific receptor RANK is expressed on hematopoietic osteoclast precursors [14], [15]. OPG also produced by osteoblast lineage cells acts as a decoy receptor and inhibits osteoclast formation, function and survival by preventing the binding of RANKL to its receptor RANK [10]. Recent data strongly revealed the significant involvement of the RANKL/RANK/OPG system in metastatic bone cancer diseases, including prostate cancer bone metastases [16]. Indeed, current studies have disclosed that blocking RANKL–RANK interaction prevents the progression of prostate cancer in bone [6], [7], [8], [9]. Furthermore, an increased expression of OPG and RANKL were reported in prostate cancer bone metastases [17], [18]. As OPG is also a decoy receptor for TNF-related apoptosis-inducing ligand (TRAIL), it exerts inhibitory effect on TRAIL-induced cancer cell apoptosis and OPG thus represents a survival factor for prostate cancer cells [19]. RANK expressed on osteoclasts/osteoclast precursors has been largely described as a key receptor that control osteoclast differentiation, activity and survival [12], [14], [15], [20]. However, we and others have demonstrated the expression of functional RANK at the surface of tumor cells that develop in bone, including a mouse osteosarcoma cell line, POS-1 [21], [22]. These recent findings bring new insights in the vicious cycle theory between bone resorbing cells and cancer cells [23]. Indeed, it has been suggested that cancer cells produce soluble factors that activate directly (RANKL) or indirectly via osteoblasts (Parathyroid Hormone-related Protein, Interleukin 8, etc.) osteoclast differentiation and maturation [24]. During the bone resorption, osteoclasts release growth factors stocked in the mineralized bone matrix (Insulin-like Growth Factor-1, Transforming Growth Factor-β, Fibroblast Growth Factor, etc.) that can further activate cancer cell proliferation [25]. This vicious cycle has been proposed to explain the tumor development in bone sites.
Previous studies reported the OPG expression by prostate cancer cells [8] and more recently RANK expression by cancer cells [21], [22], both these findings therefore led us to elucidate the molecular mechanisms underlying prostate cancer bone metastases and the therapeutic relevance of RANKL/RANK/OPG triad in these processes, using DU145 and PC3 human prostate cancer cell lines.
Section snippets
Cell culture
Human prostate cancer cell lines (DU145 and PC3) were obtained from the American Type Culture Collection (ATCC, USA). MC3T3-E1, a mouse calvaria-derived pre-osteoblast cell line was obtained from the RIKEN Cell Bank (Tsukuba, Japan). DU145 and MC3T3-E1 were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Bio Whittaker, Verviers, Belgium) and PC3 in F12 medium (Invitrogen, Eragny, France), respectively supplemented with 10% fetal bovine serum (FBS, Hyclone, France), at 37 °C in a
Human prostate cancer cell lines DU145 and PC3 express RANK and OPG
The expression of the molecular triad RANKL/RANK/OPG was analyzed in both DU145 and PC3 human prostate cancer cell lines. RT-PCR analyses revealed that both cell lines were positive for RANK and OPG but negative for RANKL at the transcript level (Fig. 1). We further analyzed the expression of major cytokines involved in tumor-bone cells cross-talks through STAT3 signal transduction pathway. Both cell lines were positive for IL-6 and LIF and negative for IL-11 and OSM at the transcript level (
Discussion
The present study was designed to better characterize the involvement of the RANKL–RANK axis in the regulation of bone cells–prostate cancer cells interactions. First, we demonstrated the functional RANK expression at the surface of DU145 and PC3 human prostate cancer cells. The functionality of this receptor was further evidenced as the shRANKL administration promoted human prostate cancer cell invasion in vitro, which was the result of the increased cell migration rather than the increased
Acknowledgments
This work was supported by INSERM, The Région des Pays de la Loire and by a grant from the West Committee of the Ligue Contre le Cancer. Kanji Mori received a personal fellowship from the Ligue Nationale Contre le Cancer.
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